Programmed necrosis is a morphologically and molecularly distinct form of cell death from apoptosis. Cell death by necrosis releases endogenous adjuvants or "danger signals" into the tissue milieu, which can trigger inflammation and stimulate immune responses. A role for programmed necrosis in anti-viral immunity is supported by recent discoveries of viral inhibitors that block this pathway of cell death. Mechanistically, programmed necrosis requires the serine/threonine protein kinas RIP1 and is optimally induced when caspases are inhibited. These results highlight a possible role for programmed necrosis in host defense against infectious agents. In order to further understand the molecular mechanism that regulates programmed necrosis, we screened by small interference RNA (siRNA) kinases and cancer-related genes that may participate in this non-apoptotic cell death pathway. We recently described the critical role of another RIP family member identified from the screen, RIP3, in TNF-induced and virus-induced programmed necrosis. In addition to RIP3, protein kinase A (PKA) catalytic subunit b and the tumor suppressor cylindromatosis (CYLD) were also identified from the RNAi screen as crucial for programmed necrosis. In this application, we will examine the molecular mechanisms by which PKA-C2 and CYLD regulates RIP1/RIP3-dependent programmed necrosis. We will examine their recruitment to the pro-necrotic signaling complex and how they might regulate the formation of the pro-necrotic signaling complex. We will determine whether deubiquitinase activity of CYLD is required for its function and explore the possible substrates for CYLD during programmed necrosis. Finally, we will test the physiological relevance of PKA-C2 and CYLD in virus infections using in vitro vaccinia virus infection as a model. PUBLIC HEALTH RELEVANCE: Cell death by necrosis causes inflammation and can greatly impact the quality of an immune response. However, the signals that regulate this process are poorly understood. We have recently identified two molecules, PKA-C2 and CYLD, to be essential for necrotic cell death. In this proposal, we will elucidate the mechanisms by which these two molecules control cell death by necrosis. Furthermore, we will evaluate how these two molecules may affect the cell death response during virus infections. These studies will allow us to better understand the signals that control cell death and inflammation. Eventually, the knowledge gained from these studies will aid the development of strategies to control inflammatory diseases caused by cell injury.